Hepatic lipase (HL) is an endothelial bound enzyme which mediates the hydrolysis of triglycerides and phospholipids present in IDL and HDL and thus, plays a central role in normal lipoprotein metabolism. Patients with HL deficiency may be at increased risk for the development of premature cardiovascular disease. In order to evaluate the feasibility of replacing an endothelial bound lipolytic enzyme in an animal model for HL deficiency, we have generated a recombinant adenovirus vector containing the human HL cDNA and the CMV- promoter (r-HL-AdV). HL deficient (HL-Def) mice were injected IV with either ten to the ninth pfu of r-HL-AdV (N=6) or control vehicle (N=3). Pre-infusion lipid values (mg/dl: TC=146plus/minus29, PL=283plus/minus56, TG=70plus/minus34, CE=80plus/minus41, FC=66plus/minus33) showed increased TC (NL=101plus/minus8, pless than 0.001), PL (NL=181plus/minus15, p<0.001), and FC (NL=35plus/minus3, pless than 0.005) when compared to age/sex matched normal mice (NL, N=13); FPLC profile of HL-Def mice demonstrated a marked increase in HDL-C and PL content relative to control mice. Immunoblot analysis of mouse post-heparin plasma 4 days post-infusion of r-HL-AdV demonstrated the presence of normal sized-HL. Virtually all expressed HL was detected in post-heparin plasma with peak activity levels of 9,193 nmoles/min/ml. HL-Def mice injected with r-HL- AdV had a peak (day 4- 5) reduction in TC (-23%, pless than 0.001), PL (-30%, pless than 0.002) and FC (-35%, pless than 0.05) whereas animals injected with control vehicle had no lipid changes. Plasma analysis on FPLC showed dramatic decreases in HDL-C and PL as well as the formation LDL-sized cholesterol and phospholipid enriched particles in r-HL-AdV treated mice. Thus, gene transfer using recombinant adenovirus resulted in successful replacement of HL in HL-deficient mice as well as normalization of the HL-deficient lipoprotein phenotype.